Redox thermodynamics of the native and alkaline forms of eukaryotic and bacterial class I cytochromes c

The reduction potentials of beef heart cytochrome c and cytochromes c(2) from Rhodopseudomonas palustris, Rhodobacter sphaeroides, and Rhodobacter capsulatus were measured through direct electrochemistry at a surface-modified gold electrode as a function of temperature in nonisothermal experiments carried out at neutral and alkaline pH values. The thermodynamic parameters for protein reduction (Delta S(rc)degrees and Delta H(rc)degrees) were determined for the native and alkaline conformers. Enthalpy and entropy terms underlying species-dependent differences in E degrees and pH-and temperature-induced E degrees changes for a given cytochrome were analyzed. The difference of about +0.1 V in E degrees between cytochromes c(2) and the eukaryotic species can be separated into an enthalpic term (-Delta Delta H(rc)degrees/F) of +0.130 V and an entropic term (T Delta Delta S(rc)degrees/F) of -0.040 V. Hence, the higher potential of the bacterial species appears to be determined entirely by a greater enthalpic stabilization of the reduced state. Analogously, the much lower potential of the alkaline conformer(s) as compared to the native species is by far enthalpic in origin for both protein families, and is largely determined by the substitution of Met for Lys in axial heme ligation. Instead, the biphasic E degrees/temperature profile for the native cytochromes is due to a difference in reduction entropy between the conformers at low and high temperatures. Temperature-dependent H-1 NMR experiments suggest that the temperature-induced transition also involves a change in orientation of the axial methionine ligand with respect to the heme plane.